Hypomethylation

Some newly introduced pyrimidine analogs (e.g., azacitidine and decitabine) differ in their mechanism of action to such a degree from the other antimetabolites that they are subgrouped under the heading DNA de- or hypomethylating agents (see below). 

Decitabine (5-aza-deoxycytosine) is an analog of the nucleoside 2 -deoxycytidine. It is believed to exert its antineoplastic effects after phosphorylation and direct incorporation into DNA and by inhibition of the enzyme DNA methyltransferase, causing hypomethylation of DNA and cellular differentiation or apoptosis. DNA hypomethylation is achieved at concentrations below those required to significantly inhibit DNA synthesis, which may promote restoration of function to genes associated with control of cellular differentiation and proliferation. Cytotoxicity in rapidly dividing cells may also result from covalent adducts between DNA methyltransferase and decitabine. 

Azacitidine, a cytidine analog, causes hypomethylation of DNA, which normalizes the function of genes that control cell differentiation to promote normal cell maturation. The suspension is administered as a subcutaneous injection daily for 7 days for the treatment of myelodysplastic syndrome, a preleukemia disease. The pharmacokinetics of azacitidine are best described by a two-compartment model, with a terminal half life of 3.4 to 6.2 hours, whereas peak concentrations are achieved 30 minutes after a subcutaneous injection.7 Azacitidine has been shown to be clinically active in the treatment of myelodysplastic syndromes. The side effects include myelosuppression, renal tubular acidosis, renal dysfunction, and injection-site reactions. 

Hypomethylation, as judged by loss of 5-methyl deoxycytidine content from DNA, occurred shortly after BaP exposure and continued upon termination of exposure conditions. Onset and persistence of hypomethylation were correlated with other types of DNA-damaging events, including strand breaks and DNA adduct formation... 

Azacytidine (Vidaza ) Hypomethylating agent Myelodysplastic syndrome Phase III... 

Luger K, Mader, AW., Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389 251-260 Ma Y, Jacobs SB, Jackson-Grusby L, Mastrangelo MA, Torres-Betancourt JA, Jaenisch R, Rasmussen TP (2005) DNA CpG hypomethylation induces heterochromatin reorganization involving the histone variant macroH2A. J Cell Sci 118 1607-1616... 

DIMS was identified as the responsible gene for one of DNA hypomethylation mutants in Neurospora crassa (Tamaru and Selker, 2001). This is the first report that histone (H3K9) methylation regulates DNA methylation. Furthermore, it is reported that this regulation is mediated through the HPl recruitment to the tri-methylated H3K9 loci (Freitag et al, 2004), but it remains to be elucidated whether DNA methylation (DNA methyltransferase) is directly controlled (recruited) by HPl. 

These findings suggest that dopamine deficiencies arising from MB-COMT promoter hypomethylation (i.e., increased expression) may influence promoter methylation and expression of RELN, DRD2, DRDl, and potentially other genes via coordinated epigenetic events that may aggravate disease symptoms (see Fig. 9.1). 

Figure 21.19 Development of a secondary carcinoma from a normal epithelium by effects of activated genes, i.e. oncogenes, and inactive tumour suppressor genes. It is somatic mutations in four or five genes in a given cell plus hypomethylation changes in histones and chromatin stracture that are involved. It is the accumulation of these genetic alterations, not the sequence, that determines the progression to a tumour cell.

The mammalian inactive X-chromosomes in females is associated with H3 methylated at Lys-9 but does not have H3 methylated at Lys-4 [165,166]. Deletion of murine Suv39h genes does not affect H3 Lys-9 methylation at the inactive X-chromosome. Thus, different histone methyltransferases are involved in the methylation of H3 Lys-9 in constitutive and facultative heterochromatin [166]. Further, HPl is not associated with the inactive X-chromosome. Methylation of H3 Lys-9 is an early event in X inactivation in mammals [167,168]. The H3 Lys-9 methylation occurs approximately at the same time as H3 Lys-9 hypoacetylation and Lys-4 hypomethylation, and happens before transcriptional inactivation of the X-linked genes. Upstream of the Xist gene, which codes for a strictly nuclear RNA involved in X inactivation, is a constitutive hotspot of H3 methylated at Lys-9. This early event of H3 Lys-9 methylation occurs simultaneously with or immediately following the association of Xist RNA with the X-chromosome. It has been proposed that the hotspot upstream of the Xist gene serves as a nucleation site for the spreading of Xist RNA and methylated Lys-9 H3 [167]. 

Dnmtl Maintenance (functions after replication on hemimethylated DNA to restore existing methylation patterns in vitro can methylate non-methylated DNA) associates with histone deacetylase Adult/Embryo DNMTD die in utero DNA in these embryos is hypomethylated, imprinted genes are biallelically expressed ES cells from DNMTl mice are viable and capable of de novo methylation... 

Again, Eu-labeled secondary antibodies are used, for example in an in vitro assay for the PKMT G9a [58] or for cellular hypomethylation of the target of the lysine methyl-transferase S ET7/9 [59]. An alternative is a radioactive assay, in which the biotinylated synthetic peptide substrate is bound to the cavities of avidin-coated microplates. 

The covalent trapping of the enzyme leads to a depletion of the cellular pool of DNMTs and subsequent DNA hypomethylation. This in turn results in activation with respect to the reactivation of silenced genes. Additionally, the covalently trapped DNMT may inhibit RNA and DNA polymerases, which leads to an inhibition of protein biosynthesis and DNA strand breaks. This may lead to apoptosis and hence cytotoxicity. Thus, it is not easy to dissect the reasons for the clinical efficacy of these inhibitors in terms of real epigenetic and plain cytotoxic effects [81]. 

Hydralazine is a vasodilating drug and inhibition of DNA methyltransferases in the range of 10-20 pM have been reported in vitro [83]. In addition, hypomethylation in cell culture has also been shovm [84]. Also the local anesthetic procaine [85] and its amide analog procainamide [84] have been identified as DNA methyltransferase inhibitors. Synthetic analogs of procaine have shown activity only around 500 pM [86] (Figure 8.10). 

Dunn, B.K. (2003) Hypomethylation one side of a larger picture. Annals of the New York Academy of Sciences,... 

Clawson GA, MacDonald JR, Woo CH. 1987. Early hypomethylation of 2 -0- Ribose moieties in hepatocyte cytoplasmic ribosomal RNA underlies the protein synthetic defect produced by CCK J Cell Biol 105 705-711. 

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